Abstract
Porcine endogenous retrovirus (PERV) is ubiquitous in pigs. The virus has drawn much attention recently because of the widespread use of pig organs and tissues in xenotransplantion. The virus has been found to be capable of infecting a bread range of cells including those of human origins in vitro and thus possesses a potential threat to xeno-transplant recipients who are generally immunosuppressed. Nevertheless, it remains unclear whether PERVs infection can cause human diseases. Therefore, the development of a highly sensitive and specific immunoassay for clinical surveillance is imperative in patients receiving xenotransplantation. We describe here the generation of monoclonal antibodies (mAbs) named A-11, 8E10, and 7C4 that specifically recognize either Gag or Env protein of PERV. In immunoblotting assay, the A-11 mAb was found to be able to detect all three classes of PERV: 8E10 mAb recognizes PERV-A and -B, whereas 7C4 mAb recognized only PERV-A. A-11 mAb can be used in detection of PERV in cultured cells, especially human kidney epithelial cells, larynx epithelial cells, hepatoma epithelial cells, and muscle spindle cells by immunochemistry. No cross-reaction with Gag and Env proteins of murine leukemia virus and human immunodeficiency virus-1,2 was observed, indicating that it was highly specific to PERV. In addition, the region recognized by mAb A-11, 8E10, and 7C4 was localized to amino acid positions 313 to 322 on the Gag protein, 427 to 434 and 517 to 537 on the Env protein, respectively. The synthetic peptides Gag313-322, Env417-434, recombinant Env397-481, and Env460-539 proteins effectively competed the binding of the mAb with recombinant Gag and Env proteins, respectively. The recombinant protein of Env was able to neutralize PERV infection. Furthermore, A-11 mAb was used in tissue tropism of PERV infection for different human cell lines by flow cytometry. Higher efficiency of PERV infection and higher expression of viral proteins were observed in human kidney cell line than others human cell lines. Therefore, these mAbs can be used as tools for the identification of viral proteins in basic research as well as clinical studies and application in neutralization of virus infection.